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Generation and functional analysis of monoclonal antibodies against the second extracellular loop of human M3 muscarinic acetylcholine receptor

机译:抗人M3毒蕈碱型乙酰胆碱受体第二个胞外环的单克隆抗体的产生和功能分析

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The M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the activation of salivary and lachrymal glands. The M3R contains four extracellular domains (the N-terminal, and the first, second, and third extracellular loops), and we recently detected antibodies against each of these four domains in a subgroup of patients with Sj?gren's syndrome (SS). Functional analysis indicated that the influence of such anti-M3R antibodies on salivary secretion might differ based on the epitopes to which they bind. To clarify the relationship between B-cell epitopes on the M3R and its function, we generated two hybridomas producing anti-M3R monoclonal antibodies against the second extracellular loop of M3R (anti-M3R 2nd mAbs) and analyzed their function by Ca 2+-influx assays, using a human salivary gland (HSG) cell line. These two anti-M3R 2nd mAbs suppressed Ca 2+-influx in the HSG cells induced by cevimeline stimulation, suggesting that autoantibodies against the second extracellular loop of M3R could be involved in salivary dysfunction in patients with SS.
机译:M3毒蕈碱型乙酰胆碱受体(M3R)在唾液和泪腺的激活中起关键作用。 M3R包含四个胞外域(N末端,第一个,第二个和第三个胞外环),最近我们在患有干燥综合征(SS)的患者亚组中检测到针对这四个域的抗体。功能分析表明,此类抗M3R抗体对唾液分泌的影响可能因它们所结合的表位而异。为了阐明M3R上B细胞表位与其功能之间的关系,我们产生了两个杂交瘤,它们产生针对M3R第二个细胞外环的抗M3R单克隆抗体(抗M3R 2nd单抗),并通过Ca 2+流入对其功能进行了分析。使用人类唾液腺(HSG)细胞系进行检测。这两种抗M3R 2nd mAb抑制了由Cevimeline刺激诱导的HSG细胞中的Ca 2+内流,这表明针对M3R第二个细胞外环的自身抗体可能与SS患者的唾液功能障碍有关。

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